Steroid receptors, either in the cell or when extracted from mammalian tissues are bound to other proteins. In this state they are referred to as being nontransformed or nonactivated. Quite recently, several groups working with the antiprogestin RU486 have suggested that receptor associated proteins are implicated in the mechanism of hormone action by acting as anti-receptors. Two receptor associated proteins have been identified. One is a heatshock protein (hsp 90). The second, p59, has not been completely characterized. Over the course of several years we have established that p59 is part of the non-transformed steroid receptor from both humans and rabbits. This has been confirmed by others (see letters from Drs. Renoir and Pratt). Furthermore, p59 is localized in the nucleus in several rabbit or monkey tissues, tissues which have been shown to contain progestin receptors. The objective of this project is to characterize p59. Key to detection and analysis of p59 is the use of a monoclonal antibody secreted by cell line KN382/ECl, an antibody produced by our laboratory. Thus to attain our goal we must produce sufficient KN382/ECl antibody to supply ourselves and other workers to allow characterization of the antigen. We shall accomplish our goals by growing KN382/ECl hybridomas in tissue culture and ascites. The purified monoclonal antibodies will be used by ourselves and others to study p59 by immunological methods, and following immunoaffinity chromatography to determine the amino acid sequence of p59. Furthermore our collaborators (see letter from Professor E.-E. Baulieu) will isolate the gene for p59. Continuing studies at The Medical College of Ohio will include recombination of isolated subunits, study of the effects of anti-hormones on the nontransformed receptors and determination of the relationship of p59to progestin receptors. This shall be accomplished by the use of SDS polyacrylamide electrophoresis, immunoblotting, high performance liquid chromatography and other assorted bio-biochemical means.